Anti cytochrome c antibody

Cells were fixed in 4% paraformaldehyde for 15 min and incubated with 0.1% Triton X-100 (Beyotime, China) for 30 min. Additionally, tumor tissues were fixed in 4% paraformaldehyde, embedded with paraffin and cut into 5-µm sections. Then, the sections were incubated with goat serum to block nonspecific binding. The sections were subsequently incubated with anti-Ki67 antibody (1:50, Proteintech, China) or anti-Cytochrome C antibody (1:100, proteintech, China) overnight at 4 °C. After washing thrice with PBS, the sections were incubated with Cy3 goat anti-rabbit IgG (1:200, Beyotime, China) and counterstained with DAPI (Biosharp, China). The results were analyzed under a fluorescence microscope.

The cells were harvested at the indicated time points. Total protein was extracted and its concentration was determined using a BCA Kit (Pierce, Rockford, IL) according to the manufacturer’s protocol. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). After being blocked with 5% nonfat milk, the membranes were incubated with rabbit polyclonal anti-NMDAR antibody (Abcam, Cambridge, Massachusetts, 1∶500), anti-Bak antibody (Abcam, 1∶1000), anti-Bax antibody (Abcam, 1∶1000), anti-cytochrome c antibody (Proteintech, Chicago, Illinois, 1∶500), or rabbit anti-β-actin antibody (Sigma, 1∶0000) at 37°C for 2 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1∶5000, Sigma). Protein expression was visualized using Odyssey V 3.0 image scanning software (LI-COR, Lincoln, NE). Semi-quantification of the protein concentrations was accomplished on the basis of three independently performed experiments. The densitometric intensities of the protein bands were quantified using Bandscan 5.0 software, and the values were normalized against β-actin for each sample.